Background: Sepsis development in patients with trauma is associated with bad prognosis. This study investigated the effect of immunomodulatory interventions in major trauma patients at high risk for sepsis.Methods: In a randomized, double-blinded, controlled design, severe trauma patients were stratified by leukocyte anti-sedimentation rate (LAR) test into high risk (HR) and low risk (LR) for sepsis. The HR patients were randomly allocated into intravenous vitamin C plus vitamin B1 (HR-CB), intramuscular vitamin D plus oral Lactobacillus probiotics (HR-DP), or control (HR-C) groups. The clinical trial was registered at clinicaltrials.gov .Outcomes: The primary outcome was Acute Physiologic Assessment and Chronic Health Evaluation score II (APACHE II) score. Secondary outcomes included sepsis incidence, changes in Sequential Organ Failure Assessment (SOFA) score, and serum monocyte chemoattractant protein-1 (MCP-1) on day 6 from baseline, 28-day mortality, intensive care unit (ICU), and hospital discharge.Results: The HR-DP, HR-CB, and LR groups showed a significantly lower incidence of sepsis development . The three groups also showed a significant improvement in APACHE II and SOFA scores. Besides, MCP-1 levels were significantly decreased in HR-DP and HR-CB groups compared to the HR-C group . Significantly decreased mortality (10% and 16% versus 60% in the HR-C group) and increased ICU discharge (95% and 84% versus 45% in the HR-C group) were observed in HR-CB and LR groups .Conclusion: Both combinations of interventions improved APACHE II scores and reduced sepsis incidence in trauma patients. The LAR combined with injury severity score were good sepsis predictors. Sepsis is a life-threatening illness associated with poor prognosis . PatientThe prevention of sepsis in patients with trauma could greatly help avoid the poor prognosis of sepsis and improve patient survival . The ideFirst, the early prediction of sepsis in trauma patients is likely to face many challenges. The surviving sepsis campaign in 2016 defined sepsis as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Organ dysfunction is identified as acute change in the total Sequential Organ Failure Assessment Score (SOFA) score ≥2 points . On applIdentifying patients at high risk for sepsis before reaching multi-organ failure was never mentioned in the surviving sepsis campaign’s latest guidelines . On the The limitation of the 2016 sepsis guidelines was addressed in the following 2021 surviving sepsis campaign’s guidelines that recommended implementing sepsis performance improvement programs in healthcare settings. These programs consist mainly of two arms: sepsis screening tools and standard operating procedures. Sepsis screening tools were defined as means of identifying high-risk critically ill patients to allow timely interventions that help improve their prognosis. Standard operating procedures involved usual care by obtaining cultures and administering fluids and antibiotics . Two metThe LAR test offered some advantages that made its use more feasible and affordable in this trial for prediction of sepsis than the MCP-1. These advantages include the performance of LAR using whole blood samples; no storage, preparation, or isolation procedures could cause false activation of leukocytes. Moreover, the LAR test is cheap, reproducible, easy to perform, and time-saving . ConversSecond, for sepsis prevention in patients with trauma at high risk for sepsis development, using a combination of interventions was recommended. The rationale behind this recommendation was the complex pathophysiology of sepsis involving hundreds of mediators and the failure of previous studies using single intervention targeting a single biomarker . SeveralVitamin D and probiotics have been used separately in the previous trials focusing on patients with trauma. The reported positive outcomes included reduced incidence of sepsis with synbiotics , signifiPrevious studies on immunomodulatory interventions in trauma usually monitored the change in interleukin 6 (IL-6) as a proinflammatory cytokine . HoweverTherefore, the aims of the current study were, first, to re-validate LAR as a cheap and available test combined with Injury Severity Score (ISS) to predict the risk for sepsis development in major trauma ICU patients and, second, to investigate the effect of IM vitamin D3 supplementation plus oral probiotics cosupplementation versus IV vitamin C plus vitamin B1 on prevention of sepsis compared to no additional supplementation. This was based on the combined predictable anti-inflammatory and antimicrobial effects of each set of study regimens on sepsis prevention in ICU patients with major trauma at high risk for sepsis development.This was a prospective, randomized, controlled, double-blind study conducted among trauma patients at high risk for sepsis in the ICU. Data were collected from February to November 2020 in the ICUs of Mansoura University Emergency Hospital, Egypt.clinicaltrials.gov .Study procedures complied with the 1964 Declaration of Helsinki and its later amendments . ConfideInclusion criteria consisted of admission to ICU within 24 h from trauma onset with ISS ≥ 16 and age ≥ 18 years. The exclusion criteria included pregnant or breastfeeding women and immune deficient patients or patients receiving immunosuppressant drugs. Patients at high risk for sepsis (LAR < 15%) who had serum vitamin D level <10 ng/ml or >30 ng/ml or serum calcium level >10 mg/dl were excluded. Besides, patients with a history of primary parathyroid disease and those with contraindications to enteral administration were also excluded. Patients with end-stage renal disease on renal replacement therapy were not eligible for the study. Moreover, patients with oxalate nephropathy or glucose-6 phosphate dehydrogenase deficiency were also not eligible for the study.The primary outcome of the study was the change in Acute Physiologic Assessment and Chronic Health Evaluation score II (APACHE II) score defined as day 6 minus day 0 score, while the secondary outcomes included the change in SOFA score and MCP-1 in addition to number of patients who developed sepsis within the first week. Blood cultures were used as a possible documentation for infection. Moreover, C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR) were also measured on days 0 and 6 for all included patients. Additional secondary outcomes included ICU discharge, hospital discharge, and mortality within 28 days for all patients.In a secondary analysis, the predictive value of LAR combined with ISS to predict the risk for sepsis development in severe trauma ICU patients was evaluated.Sample size calculation was based on APACHE II scores achieved after receiving vitamin C, vitamin D, and probiotics in previous studies . For vitt-test was used to detect difference between two independent means (two groups), two-tailed, with α error = 0.05 and power = 89%. The effect sizes were 1.5228481, 1.1855969, and 1.1051758, whereas the total calculated sample sizes were 10, 16, and 18 patients in each arm for vitamin C, vitamin D, and probiotics, respectively. To overlap the probable dropout of patients, 10% of the calculated sizes were added, making the total calculated sample sizes of 11, 18, and 20 for vitamin C, vitamin D, and probiotics, respectively. Thus, we decided to include 20 patients in each group.G*Power version 3.0.10 was used for sample size calculation. The After ICU admission of patients with ISS ≥ 16, all patients were evaluated. The included patients with high risk for sepsis (LAR < 15%) were randomly allocated, at 1:1:1 ratio, into one of three groups each consisting of 20 patients, using sealed opaque envelopes. Patients in the first group did not receive any additional supplement and represented the control group (HR-C group). Patients in the second group received vitamin D plus probiotics (HR-DP group), while patients in the third group received vitamin C plus vitamin B1 (HR-CB group). The low-risk (LR) group (LAR ≥ 15%) did not receive any special therapy.Demographic characteristics , comorbidities, initial ventilatory status, Glasgow coma score (GCS), and laboratory values were collected on admission. The ISS determination was performed according to On day 0, recordings of APACHE II  and SOFALactobacillus probiotics  in a dose of six sachets (one pack) twice a day (at 9 a.m. and 9 p.m.) orally (either directly or through Ryle’s tube feeding) starting from day 1 for 48 h.In the HR-DP group, patients received vitamin D as one IM injection  on day 1 in addition to Patients of the HR-CB group received from day 1 a dose of 1 g of vitamin C (one ampoule of Wörwag Pharma GmbH and Co. KG® Vitamin C 1000 mg) plus 200 mg of vitamin B1 . Vitamin C plus vitamin B1 were infused intravenously in 500 ml of saline over 30 min four times at 12-h intervals for 48 h.Intradermal skin testing (IDT) for vitamin B1 hypersensitivity was conducted in patients of the HR-CB group with unspecified history of allergy to vitamin B1. Patients showing allergy to vitamin B1 were excluded from the study. Blood gases were investigated for metabolic acidosis. Patients in the HR-CB group showing metabolic acidosis on day 1 were also excluded from the study.The investigator who knew the allocation of groups and was responsible for the drug administration was excluded in all data collection.On day 6, SOFA and APACHE II scores were recorded for all groups. Moreover, a blood sample was obtained from all patients for determination of MCP-1 (in HR groups), ESR, and CRP level  measurements. Changes in SOFA and MCP-1 were defined as day 6 minus initial (day 0) values. For LR group, outcomes were the same as the other three groups except for change in MCP-1 as MCP-1 for this group was only measured on day 0. Eight centimeters of blood were collected for aerobic blood culture .During the whole ICU admission, all patients in the four groups were carefully monitored and managed according to the ICU protocol. The number of patients who developed sepsis in each group within 7 days was recorded. Sepsis development within 7 days was confirmed according to the sepsis-3 criteria . Sepsis For fear that the patient cannot complete the study , after completion of study treatment regimen, and before the patient completes day 6, a reserved blood sample and blood culture were collected on day 3. This reserved blood sample was used for MCP-1 (in HR groups), ESR, and CRP level measurements. These reserved samples and blood culture taken on day 3 were collected to be analyzed immediately (except for MCP-1), recorded if the patient did not complete the study, and discarded if day 6 blood sample and blood culture were collected. Also, for those patients, the last recorded APACHE II and SOFA scores (after day 2) were forwarded for assessment, whereas if a patient was discharged to the ward before day 6 but after completing the study regimen in the ICU, the last recorded APACHE II score in the ICU just before discharge was used. Then, the patient was followed in the ward, and the final SOFA score, ESR, CRP level, MCP-1 level (if HR group), and blood culture were collected in the ward on day 6. Patients who were unable to complete their study treatment regimens in the ICU due to very early discharge or death were excluded from the study.Serum creatinine level in the ICU was routinely monitored for any significant elevations. Moreover, the serum creatinine level on day 6 was compared to day 0 to record the occurrence of acute kidney injury (AKI). Patients with AKI were managed according to the Kidney disease Improving Global Outcomes (KDIGO) guidelines .Serum vitamin D level was assessed using the LIAISON® analyzer, DiaSorin S.p.A. The LIAISON® 25-hydroxyvitamin D assay is a direct, competitive chemiluminescent immunoassay for quantitative determination of total 25-hydroxyvitamin D in serum or plasma. This method of immunoassay is FDA approved .Blood samples (3 cm) were obtained in vacuum red cap disposable plain blood tubes  and centrifuged at 370 × g for 5 min . Serum samples were collected and stored at −80°C, analyzed together after all patient enrollments. The MCP-1 was assessed using the commercially available Invitrogen Human C-C motif chemokine ligand 2 [CCL2 (MCP-1)] ELISA kit .Two sets of the ELISA kits were used according to the manufacturer’s instructions. Each kit contained one plate [Microwell Plate (12 strips of eight wells each) coated with monoclonal antibody to human MCP-1]. Samples were diluted at 1:5 [20 μl sample +80 μl assay buffer (1×)]. The standard curve was constructed, and the MCP-1 level in each sample was retrieved from the standard curve and multiplied by the dilution factor (×5). Samples exceeding standard concentration were further externally prediluted.The ESR was measured by modified Westergren method using Streck® ESR-10 Manual Rack for the Modified Westergren Sed Rate, Streck® 240321. However, the CRP level was measured by nephelometry using the BN™ II System nephelometric analyzer.Peripheral venous blood (1.28 ml) was collected in sodium citrate anticoagulated tube . After 1 h of blood sedimentation, using an automatic cell counter , leukocyte count in the upper (U) and lower (L) half of blood column was determined. LAR was calculated according to the equation described by The IBM® SPSS® 26.0.0 statistical software was used to perform statistical analyses. Shapiro–Wilk test for normality was performed. Quantitative data were summarized as mean ± SD or median, interquartile range according to normality. Qualitative data were summarized as frequency (percentage). To detect differences between groups, analysis of variance (ANOVA), Kruskal–Wallis, and chi-square tests were used for parametric, nonparametric, and categorical variables, respectively. If significant differences between groups were found, appropriate post-hoc tests were performed. Post-hoc tests after ANOVA were determined according to homogeneity of variances. Dunn’s and Monte Carlo post-hoc tests were conducted after Kruskal–Wallis and chi-square tests, respectively.t-test and Wilcoxon signed-rank test were used for parametric and nonparametric data, respectively.To determine if there were significant differences between day 0 and 6 scores (SOFA or APACHE II) within the same group, paired Kaplan–Meier and log rank test were used to compare ICU mortality between HR groups. Cox’s proportional hazards model was used to identify significant independent predictors associated with ICU mortality with calculation of the hazard ratios and 95% confidence intervals. Univariate models were used for determining which variables could be associated with ICU mortality in HR groups (60 patients). The tested variables in the univariate model included the effect of study treatment (CB and DP interventions compared to no intervention in the HR-C group), the initial GCS (three to eight versus higher GCS), the need for vasopressors, ISS (≥25 versus lower ISS), sepsis development (by the end of the first week), and needing mechanical ventilation at admission. Only variables that showed statistical significance in univariate models were included in the multivariate model.p-value) ≤ 0.05 was considered statistically significant.Receiver operating characteristics (ROC) curve was used to evaluate the predictive ability of different sepsis predictors  among the HR-C and LR groups. Test performance for predictors was evaluated as failed , poor , fair , good , and excellent (0.9–1) . ProbabiBetween February and November 2020, 156 patients were evaluated. After ruling out patients who did not fulfill the study criteria, 112 patients were enrolled after obtaining informed consent. Then, 27 patients were excluded as they did not complete the steps of the study. Afterward, 85 patients had successfully completed the study .p-value < 0.0001). Similarly, arterial oxygen saturation and serum 25-hydroxyvitamin D level were significantly high in the LR group compared to high-risk groups with p-values of 0.002 and <0.0001, respectively.Patients’ demographic data and initial ventilatory status  showed nThere were no significant differences between the groups with respect to ISS, cause of trauma, and primary diagnosis (type of trauma), even in the segmental injury description. Intracranial hematoma (≤100 ml or unspecified) represented the most prevalent injury in all patient groups either isolated or combined with other traumas , 4.p-value < 0.0001). On day 6, a significant decrease was detected in both HR-CB and HR-DP groups compared to the HR-C group .p-value = 0.014). Interestingly, both HR-DP and HR-CB groups showed a significant decrease in MCP-1 level on day 6 compared to that on day 0 .Comparing the serum MCP-1 levels within the same group, the HR-C group showed a significant increase in MCP-1 level on day 6 compared to day 0 .The ESR, at the first and second hours, revealed no significant differences between groups. Within the same group, the ESR showed a significant increase in the HR-DP and HR-C groups on day 6 compared to that on day 0 at both the first and second hours . Within the same group, the HR-CB group showed a significant decrease in serum CRP level on day 6 compared to that on day 0 .The serum CRP level on day 6 revealed a significant decrease in both the LR and HR-CB groups compared to that in the HR-C group . Noteworthily, the LR, HR-DP, and HR-CB groups showed a significant improvement in APACHE II score on day 6 compared to their initial score on day 0  and a significant improvement compared to the HR-C group on day 6 (Monitoring the improvement (decrease) or deterioration (increase) in clinical and laboratory items of APACHE II score on day 6 compared to those on day 0 within the same group, the HR-C group showed a significant deterioration in APACHE II score , while the LR, HR-DP, and HR-CB groups showed a significant improvement in SOFA score . Furthermore, a significant improvement was observed in SOFA score of the HR-DP, HR-CB, and LR groups compared to the HR-C group on day 6 . The coagulase negative Staphylococcus aureus (CONS) represented the most abundant species isolated from positive aerobic blood cultures in all groups   .p-value = 0.001) and hospital discharge     in addit= 0.001)  comparedp-value = 0.22).To evaluate the occurrence of AKI among the study population, serum creatinine levels on day 6 were compared to the initial values on day 0. It was observed that 10 patients developed AKI. These patients were distributed as follows: two, five, one, and two patients in the LR, HR-C, HR-CB, and HR-DP groups, respectively, with no statistically significant differences  and sepsis development , whereas all the other tested variables in HR groups were non-significant . Hence, the multivariate Cox proportional hazard model was performed using effect of study treatment and sepsis development as covariates . Regarding the effect of study treatment versus control in HR groups, the HR-CB group showed the lowest hazard ratio for ICU mortality compared to the HR-C group. However, the difference between hazard ratios did not reach the threshold of statistical significance .Survival analysis showed that the HR-CB group had a significantly lower ICU mortality compared to the HR-C group . The univariates . Patientp-value = 0.001), 0.734 , and 0.758 , respectively . Areas under the ROC curve (AUCs) of MCP-1 (day 0), ISS (day 0), and 100-LAR (day 1) were 0.793  with no other complications. Consequently, these patients were excluded from the study. No other adverse events were deemed related to the study drugs in the HR-CB and HR-DP groups in the entire study period.In the current study, LAR was used for determination of patients who have high risk for sepsis development. The effects of immunomodulatory interventions  on prevention of sepsis development were investigated among patients with major trauma at high risk for sepsis. Both interventions decreased the incidence of sepsis development to the same extent (20%). However, vitamin C plus vitamin B1 were associated with lower 28-day mortality rate and higher ICU and hospital discharge rates than vitamin D plus probiotics.Lactobacillus fermentum, one of the components of probiotic product used in this study, is among the most studied Lactobacilli strains with antimicrobial activity (This current study showed that vitamin D plus probiotics significantly decreased scores for illness severity (APACHE II and SOFA), proinflammatory biomarker MCP-1, and sepsis development. The overall good clinical outcomes observed in the HR-DP group may be attributed to the synergistic effects of vitamin D plus probiotic combination. The benefits of vitamin D and probiotic cosupplementation on inflammation and antioxidant capacity have been studied in other contexts than ICU severe trauma . Lactobaactivity . The antactivity . Probiotactivity . Bacteriactivity . Besidesactivity . The antactivity . Both hiactivity .via inhibition of nuclear factor kappa B signaling, antioxidant potential, and mitochondrial protective mechanisms  as a predictor of sepsis is concordant with a previous study reporting good test performance of LAR  as a predictor of bacteremia in a general surgical ICU population . FurtherThe significant decrease in SOFA score and consequently the significantly lower incidence of sepsis among the intervention groups compared to control were concordant with previous studies conducted on the use of synbiotics , vitaminBedreag et al. found no reduced incidence of sepsis development with the use of vitamin C, vitamin B1, and N-acetyl cysteine together among ICU trauma patients . HoweverIf any patient was discharged to the ward before day 6 after completing the study treatment regimen in the ICU, final SOFA score and blood culture were collected in the ward on day 6. However, the last APACHE II score in the ICU just before discharge was recorded and forwarded for assessment. This is based on evidence from literature that full SOFA score is the best tool for identifying patients with sepsis in the ward setting (better than quick SOFA) . HoweverAll three groups  revealed a significantly lower incidence of sepsis than the HR-C group by the end of the first week. Consequently, these three groups showed lower 28-day mortality than the HR-C group. Multivariate Cox regression showed that sepsis development was a significant risk factor for ICU mortality in HR groups. These results comply with a previous study reporting sepsis as a leading cause of mortality contributing to 11 million deaths annually worldwide . AnotherDifferently from this study, previous studies investigating the effects of interventions (CB and DP) on MCP-1 levels were either preclinical or clinical on patients without trauma . The sigin vitro study. The 1,25 dihydroxyvitamin D concentration used in Alvarez et al. ’s in vitro study (16 ng/ml) was in the range of 25-hyroxivitamin D levels of patients in the HR-DP group (10–30 ng/ml)  . The eff0 ng/ml) . In the 0 ng/ml) . Anotherof MCP-1 . Lactobais study . The resA significant increase in ESR and a nonsignificant increase in CRP level were found within both HR-DP and HR-C groups on day 6 compared to those on day 0. However, in the HR-CB group, a nonsignificant increase in ESR besides a significant reduction in CRP level were observed on day 6 compared to those on day 0. These ESR and CRP changes in HR groups agreed with previous studies reporting the higher sensitivity of CRP to changes in acute phase response than ESR . KotzampControversially, The CRP level of the HR-CB group on day 6 was significantly lower compared to that in the control group. This result was concordant with the previous study of Sandesc et al. who found a significant decrease in CRP level and ESR in the intervention group compared to those in the control group upon ICU discharge .The ESR showed an increase that was significant within the HR-DP group and insignificant within the HR-CB group on day 6 compared to that on day 0. One explanation is that fibrinogen and immunoglobulin G are the main proteins influencing ESR. Both fibrinogen and immunoglobulin G have long half-lives . Thus, eAerobic bacterial blood cultures were used as a possible documentation for infection due to their prevalence in sepsis diagnosis among the critically ill rather than anaerobic bacteria, fungi, or viruses . The mosPatients in the HR-CB group mechanically ventilated from day 0 showed a significantly shorter duration of mechanical ventilation compared to those in the HR-C group by the end of the first week. These findings were discordant with the results of Sandesc et al., who attributed the nonsignificant difference in duration of mechanical ventilation between their groups to the high prevalence of thoracic trauma and pulmonary infections . HoweverAt the end of the first week, the occurrence of AKI detected by comparing day 6 and day 0 serum creatinine values followed the KDIGO guidelines. The KDIGO guidelines define the AKI as an increase in serum creatinine level to 1.5 times the baseline creatinine or more within the last 7 days . AKI is One strength for this study is that, to the best of our knowledge, it is the first study to demonstrate the lowering effects of vitamin C plus vitamin B1 (CB) and vitamin D plus probiotic (DP) combinations on MCP-1 in ICU trauma patients. The average estimated serum vitamin C level in this trial (0.4 mM) was far from the level reported in a previous preclinical study to be associated with prooxidant effects (2 mM) . BesidesThis study may be limited by the inability to measure vitamin C and vitamin B1 levels at baseline due to the requirement of high-performance liquid chromatography (HPLC), which was expensive and unavailable. Moreover, HPLC may be unable to detect the very low levels of vitamin C in critical illness . VitaminEarly prediction of sepsis in severe trauma represents an unmet clinical need. The use of LAR as a point-of-care test combined with ISS as a cheap and available alternative to MCP-1 plus ISS enabled determination of patients at high risk for sepsis development who would benefit most from the prophylactic immunomodulatory interventions. Vitamin D plus probiotics synergistic combination reduced the incidence of sepsis development similar to IV vitamin C plus vitamin B1 in the ICU patients with trauma. Both combinations reduced MCP-1 level, proving the therapeutic progress accompanying MCP-1 level decrease in severe trauma. Hence, the administration of immunomodulatory interventions for prevention of sepsis in clinical practice could help improve major trauma patient prognosis and decrease the incidence of sepsis.


Non-small cell lung cancer (NSCLC) is a malignant tumor with the highest mortality rate in our country. It has been found in many studies that microRNA-4521 (miR-4521) is abnormally expressed and plays a role in clear cell renal cell carcinoma and other cancers.The purpose of this study was to explore the relationship between miR-4521 expression and clinical prognosis, as well as its influence on cell biological behavior.The expression differences of miR-4521 in NSCLC tissues and cells were examined by qRT-PCR technology. Kaplan–Meier survival analysis and Cox regression analysis were used to analyze the clinical information and survival status of patients to explore the relationship. Using the vitro cell MTT assay, Transwell assay, and western-blot analysis, the effects of miR-4521 on cell proliferation, migration, and invasion were analyzed.The expression of miR-4521 in NSCLC tissues and cells was significantly downregulated. miR-4521 can be used as an independent prognostic factor. The survival rate of the miR-4521 low expression group was lower, which was significantly related to poor prognosis. In addition, the low expression of miR-4521 significantly promoted cell proliferation, migration, and invasion with highly expressed related protein levels. FOXM1 might be a direct target of miR-4521.The results of this study showed that the low expression of miR-4521 indicated the poor prognosis of NSCLC and promoted cell proliferation, migration, and invasion by targeting FOXM1. Non-smaMicroRNAs (miRNAs) are small noncoding RNAs with a length of about 17–22 nucleotides . miRNAs in vitro cell experiments. This study may provide some theoretical support for the development of prognostic biomarkers for NSCLC patients.In this study, we examined the abnormal expression of miR-4521 in NSCLC tissues and cells. Then, we analyzed the correlation of its expression with clinical characteristics and survival status to understand its clinical significance. We also further explored its role in cell activities through 22.1We selected 131 patients who underwent NSCLC surgical resection in China–Japan Union Hospital of Jilin University between January 2013 and December 2014. The patients did not receive any other adjuvant treatments before surgery. Their clinical conditions are shown in 2.22.Human NSCLC cell lines  and human normal lung epithelial cell line BEAS-2B were purchased from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium  supplemented with 10% FBS  in a humid 37°C incubator containing 5% CO2.3First, the cells were cultured in six-well plates. Then, we used Lipofectamine 2000  to transfect miR-4521 mimic, mimic negative control (NC), miR-4521 inhibitor, or inhibitor NC  into the cells. The untreated cells were used as a blank control. All cells were quantified by qRT-PCR technology after transfecting.2.4Ct−ΔΔ method.We used TRIzol reagent  to extract RNA from tissues and cell lines, and then used NanoDrop to determine the concentration of the extract to quantify RNA. Next, we used miRNA first-strand cDNA synthesis kit  to reverse transcribe miRNA. According to the manufacturer’s instructions, we performed qRT-PCR on the 7900 real-time PCR system  and used SYBR Green  to detect the miR-4521 expression in the tissues and cell lines. U6 was selected as the endogenous control and the relative value of its expression level was calculated by the 22.54 cells/well) were cultured in a 96-well plate and an MTT reagent  was added to each well at 0, 24, 48, and 72 h. After incubating for 4 h, the cell suspension was removed and dimethyl sulfoxide  was added. Finally, the absorbance was measured at 490 nm using a microplate reader.In this study, an 3--2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay was used to verify the effect of miR-4521 on cell proliferation. The transfected cells (1 × 102.64 cells/well) to the upper chamber of the Transwell. At the same time, the serum-containing medium as a chemokine in the lower chamber was also added. After culturing for 24 h at 37°C, the cells in the upper chamber were fixed in methanol for 30 min and then stained with 1% crystal violet for 15 min. Then, the number of cells were counted using a microscope.The cell migration and invasion assays were verified with the Transwell chamber , and the invasion assay required Matrigel  pre-coated on the bottom of the upper chamber. We cultured the transfected cells in a serum-free medium for a certain period and then added the cell suspension (5 × 102.7A549 cell lysates were prepared with RIPA buffer (Santa Cruz Biotechnology) containing 1% phenylmethylsulfonyl fluoride on ice and then centrifuged at 12,000 rpm at 4°C. The supernatant was collected and the protein concentrations were measured using the bicinchoninic acid method (Thermo Scientific). Lysates with equal amounts of protein were used for electrophoresis using 10% SDS-PAGE for protein separation. Then, the protein was transferred onto the poly (vinylidene fluoride) membrane  in Tris/glycine/SDS buffer and blocked in 5% milk for 1 h. The membranes were incubated with the following primary antibodies: anti-cyclin D1 antibody , anti-Cdk4 antibody , anti-MMP2 antibody , anti-MMP9 antibody , and anti-β-actin  overnight. The blots were washed with TBST and were exposed to HRP-labeled goat anti-rabbit IgG secondary antibodies  for 1 h. Protein bands were visualized by electrochemiluminescence (ECL) and scanned by the software ChemoiDox MP system .2.8Bioinformatic analysis using TargetScan 7.2 database showed that FOXM1 possesses binding sites of miR-4521 with a high context score percentile of 98. FOXM1 with miR-4521-binding sites was inserted into the pGL3 plasmid to construct the FOXM1 wild-type (WT) plasmid. Besides, the mutant miR-4521-binding sequence was synthesized to construct the FOXM1 mutant (MUT) plasmid. A549 cells (with the lowest miR-4521 expression levels) were incubated in 48-well plates for 24 h. Then, cells were transfected with FOXM1-WT or FOXM1-MUT plasmid in combination with miR-4521 mimic, inhibitor, or controls using Lipofectamine 2000 . After transfection for 48 h, a Dual-Luciferase Assay kit (Promega) was used to analyze the relative luciferase activities.2.9t-test or one-way ANOVA. Kaplan–Meier analysis and Cox regression analysis were used to determine the relationship between miR-4521 expression level and clinical characteristics of patients. All determinations were repeated more than three times, and the results are expressed as mean ± SD. P < 0.05 means significant.SPSS22.0 software  and GraphPad 7.0  were used for statistical analysis. The significant differences between different groups were analyzed by Student’s 33.1P < 0.001, P < 0.001, We first examined the expression of miR-4521 in 131 pairs of NSCLC tissues and adjacent noncancerous tissues by qRT-PCR. It can be seen from the results that the expression of miR-4521 in NSCLC tissues was significantly lower than that in adjacent noncancerous tissues (3.2n = 61) and low-expression group (n = 70). The grouping was based on the average expression of miR-4521 in the tissues of NSCLC patients. P = 0.016) and TNM staging (P = 0.011), but is not correlated with other parameters including age, gender, tumor size, and histological type .We divided NSCLC patients into high-expression group (3.3P = 0.004). The multivariate Cox risk regression model was used to analyze the relationship between the expression of miR-4521 in tissues and the survival status of the corresponding patients. It can be concluded that the expression of miR-4521 , lymph node metastasis , and TNM staging  can be used as independent factors for the prognosis of NSCLC , while the miR-4521 mimics significantly increased its expression in cells (P < 0.01) , while miR-4521 inhibitors significantly enhanced cell migration and invasion capabilities (P < 0.01) ( < 0.01) . The tra < 0.01) .3.5Cyclin D1, Cdk4, MMP2, and MMP9 are cell proliferation, migration, and invasion-related proteins. Thus, we further measured the expression of these proteins in A549 cells transfected with mimic, inhibitor, or NCs using Western blot assay. miR-4521 overexpression led to the downregulation of cyclin D1, Cdk4, MMP2, and MMP9 .3.6P < 0.05). However, the luciferase activities of cells co-transfected with FOXM1 MUT and mimics had no significant changes. Thus, we believed that FOXM1 is a downstream target of miR-4521 in NSCLC cells.To uncover the potential mechanism of the decreased expression of miR-4521 in NSCLC cells, the potential targets of miR-4521 were identified using Targetscan prediction tools. Among these targets that were predicted, Forkhead Box M1 (FOXM1) was further studied. The binding sites between FOXM1 3′-UTR and miR-4521 are shown in 4NSCLC is the major pathological type of lung cancer, with high morbidity and mortality, which is easy to relapse and difficult to be monitored . TherefomiR-4521 may be used as a tumor suppressor gene for NSCLC. Our study has found that compared with the control group, the expression of miR-4521 in NSCLC tissues and cell lines was significantly downregulated. By analyzing the relationship between the expression of miR-4521 and the clinical characteristics of patients, it can be concluded that the low expression of miR-4521 is significantly related to TNM staging and lymph node metastasis. These results are consistent with the existing research results. Pekarsky et al. have proved that miR-4521 was downregulated in tumor samples from patients with CLL . Feng XumiR-4521 may be a prognostic biomarker of NSCLC. We used the Kaplan–Meier survival curve and Cox regression model to explore the relationship between miR-4521 expression and patient prognosis. It can be concluded from the survival curve that the survival rate of patients with low expression is lower and low expression of miR-4521 indicates a poor prognosis. At the same time, the Cox regression model can prove miR-4521 is an independent prognostic factor for NSCLC. Liao et al. have found that miR-4521 showed a good prognostic ability and can be used as an independent prognostic factor for the survival of pancreatic ductal adenocarcinoma . These rin vitro migration and invasion ability of ccRCC cells 786-O and ACHN through the TIMP-1/MMP2/MMP9 pathway, and promote their apoptosis through the MDM2/p53/Bcl2/Bax pathway to reduce its proliferation [The low expression of miR-4521 promoted the proliferation, migration, and invasion of NSCLC cells. We studied the effects of miR-4521 mimics or inhibitors on the biological behavior of NSCLC cells using MTT and Transwell assays. These results showed that low expression of miR-4521 can enhance cell proliferation, migration, and invasion ability, while high expression of miR-4521 can reduce its ability. Furthermore, enhanced expression of miR-4521 decreased the proliferation-, metastasis-related protein levels, including cyclin D1, Cdk4, MMP2, and MMP9. These data suggested that miR-4521 influences A549 cell malignancy via cyclin D1, Cdk4, MMP2, and MMP9. A previous study also demonstrated that the overexpression of miR-4521 can reduce the feration . Furtherferation . FOXM1 wferation ,37. BaseThis study indicated that the expression of miR-4521 is downregulated in NSCLC tissues and cell lines, and its low expression is significantly related to the poor prognosis of patients. The low expression of miR-4521 promoted cell proliferation, migration, and invasion by targeting FOXM1. In short, miR-4521 may serve as a new prognostic biomarker and the miR-4521/FOXM1 axis may be a therapeutic target for the treatment of NSCLC patients.


We previously reported that chronic ethanol exposure from adolescence facilitated the sensory-evoked molecular layer interneuron–Purkinje cell synaptic transmission in adult mice in vivo. Herein, we investigated the effect of chronic ethanol exposure from adolescence on facial stimulation-evoked MF–GC synaptic transmission in the adult mouse cerebellar cortex using electrophysiological recording techniques and pharmacological methods. Chronic ethanol exposure from adolescence induced an enhancement of facial stimulation-evoked MF–GC synaptic transmission in the cerebellar cortex of adult mice. The application of an N-methyl-D-aspartate receptor (NMDAR) antagonist, D-APV (250 μM), induced stronger depression of facial stimulation-evoked MF–GC synaptic transmission in chronic ethanol-exposed mice compared with that in control mice. Chronic ethanol exposure-induced facilitation of facial stimulation evoked by MF–GC synaptic transmission was abolished by a selective GluN2A antagonist, PEAQX (10 μM), but was unaffected by the application of a selective GluN2B antagonist, TCN-237 (10 μM), or a type 1 metabotropic glutamate receptor blocker, JNJ16259685 (10 μM). These results indicate that chronic ethanol exposure from adolescence enhances facial stimulation-evoked MF–GC synaptic transmission via GluN2A, which suggests that chronic ethanol exposure from adolescence impairs the high-fidelity transmission capability of sensory information in the cerebellar cortex by enhancing the NMDAR-mediated components of MF–GC synaptic transmission in adult mice in vivo.Sensory information is transferred to the cerebellar cortex  Ethanol is the most widely used and abused psychoactive substance and can cause damage to the central nervous system that leads to impairment of its function. The cerebellum is a major target of ethanol. Ethanol exposure causes alterations in behavior, motor coordination, speech, balance, and cognitive functions, which are considered to be induced by the impaired function of cerebellar neuronal circuits and synaptic transmission .In vitro, acute ethanol exposure was shown to increase the frequency of miniature and spontaneous inhibitory postsynaptic currents in cerebellar Purkinje cells (PCs) and molecular layer interneurons by enhancing γ-aminobutyric acid (GABA) release in rats  responses in the mouse cerebellar molecular layer   , and sig(NMDARs) . Chronic(NMDARs) . Chronic pathway  and signr cortex .via convergent multimodal mossy fibers (MFs), increasing the use for learned associations  are the main integrators and processors of input information in the cerebellar cortex  and are ciations . Sensoryciations . Passiveciations . GCs areciations . Cerebelciations . The spiciations . GCs exhciations . In addil fibers , which ibranches . In the branches  and inhiactivity . Chronicactivity . Adolescactivity , especiaactivity . During activity . Chronicactivity  and to iactivity .Cerebellar GCs exhibit high-frequency and high-fidelity properties during sensory information encoding and transfer to PCs, which are critical to motor regulation and motor learning behavior because of the modulation of the output of PCs. Chronic ethanol exposure from the adolescent stage could damage the cerebellar function by impairing the synaptic transmission of sensory information from MFs to GCs. Therefore, in this study, we studied the effects of chronic ethanol exposure in adolescence on facial stimulation-evoked MF–GC synaptic transmission in the cerebellar cortex of mice.A total of 96 (5-week-old) ICR mice were selected, and they were divided into the chronic ethanol exposure group  and the control group . The experimental procedures were approved by the Animal Care and Use Committee of the Yanbian University and were in accordance with the animal welfare guidelines of the United States National Institutes of Health. The permit number is SYXK (Ji) 2011-006. All animals were housed under a 12-h light/dark cycle with free access to food and water in a colony room kept under a constant temperature (23 ± 1°C) and humidity (50 ± 5%). Mice in the chronic ethanol group were given intraperitoneal (i.p.) injection of ethanol , while the mice in the control group were given i.p. injection of the same volume of saline. Ethanol (95%) was diluted in saline to a final concentration of 15%. The i.p. injection was performed 1 time/day during 8:00–9:00 a.m. for 28 days. The electrophysiological recordings were performed 1 day after the last injection of ethanol.4, 2 mM CaCl2, 1 mM NaH2PO4, 25 mM NaHCO3, and 10 mM D-glucose) with a peristaltic pump  at 0.5 ml/min. Rectal temperature was monitored and maintained at 37.0 ± 0.2°C.The anesthesia and surgical procedures have been described below , b. The Local field potential recordings from the GL were performed with an Axopatch-200B amplifier . The potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.4 software (Molecular Devices). Recording pipettes were made with a puller  from a thick-walled borosilicate glass . Recording electrodes were filled with ACSF and with resistances of 3–5 MΩ. The recordings from the GL were performed at depths of 300–350 μm under the pia mater membrane.® III; Parker Hannifin Co., Pine Brook, NJ, United States). The air-puff stimuli were controlled by a personal computer, were synchronized with the electrophysiological recordings, and were delivered at 0.05 Hz via a Master 8 controller  and Clampex 10.4 software. For isolating mossy fiber-granule cell (MF-GC) synaptic transmission, picrotoxin (100 μM) was added to ACSF during all recordings to prevent GABAA receptor-mediated inhibitory responses of Golgi cells. Single stimulation  or stimuli train  were used to evoke the MF-GC synaptic transmission in the absence of GABAA receptors activity.Facial stimulation was performed by air-puff to the ipsilateral whisker pad through a 12-gauge stainless steel tube connected with a pressurized injection system  were bought from Sigma-Aldrich . PEAQX and TCN 237 were purchased from Tocris . The drugs were dissolved in ACSF and applied directly onto the cerebellar surface by a peristaltic pump (0.5 ml/min).Picrotoxin, -(cis-4-methoxy- cyclohexyl)-methanone [JNJ16259685 (JNJ)] and post hoc test) and a two-way ANOVA (SPSS software) were used to determine the level of statistical significance among the groups of data. P-values below 0.05 were considered as statistically significant.The electrophysiological data were analyzed using Clampfit 10.4 software . All data are expressed as the mean ± SEM. A one-way ANOVA (the Turkey A receptor antagonist picrotoxin (100 μM), which blocks the inhibitory components of Golgi cells. Blockade of GABAA receptor activity and air-puff stimulation of the ipsilateral whisker pad  evoked negative components N1 and N2 in the GL (n = 10 mice), which was similar to that in the control group  in the ethanol-exposed group, which was significantly larger than that in the control group , which was significantly lower than that in the control group , which was also significantly larger than that in the control group  in several regions of the brain , and actvia GluN2A, which suggests that chronic ethanol exposure from adolescence may impair high-fidelity properties during the sensory information processing in the cerebellar cortical GL.In this study, we found that the chronic ethanol exposure from adolescence led to an enhancement in the facial stimulation-evoked MF–GC synaptic transmission, which was blocked by a selective NMDAR antagonist. The chronic ethanol exposure-induced enhancement of the facial stimulation-evoked MF–GC synaptic transmission was prevented by a selective GluN2A blocker but was not prevented by a selective GluN2B antagonist or a selective mGluR1 blocker. These results indicate that the chronic ethanol exposure from adolescence enhances facial stimulation-evoked MF–GC synaptic transmission Chronic ethanol exposure can affect neurotransmitter release, synaptic transmission, and neural circuit plasticity in multiple brain regions. The latter is related to tolerance and dependence in human . Heavy cin vivo.In the cerebellar cortex, GCs receive and respond to sensory information conveyed by MFs  and GolgD-aspartate receptors are a crucial target of chronic ethanol exposure in the central nervous system and are involved in tolerance, dependence, withdrawal, craving, and relapse (N-methyl- relapse . Acute e relapse . Chronic relapse . Many st relapse  and that relapse . Chronic relapse , 2010. C relapse , 2010, tin vivo (via GluN2A but not via GluN2B. Previous studies also showed that the NR2B mRNA expression was significantly increased after chronic ethanol exposure (in vivo.In a previous study, NR2A and NR2C mRNA were detected in cerebellar GCs during the second postnatal week, whereas the NR2B mRNA was transiently expressed in the GCs during the first 2 postnatal weeks in rats . GluN2A in vivo . It has in vivo . Howeverexposure , particuexposure . NR2B orexposure . Ethanolexposure , 2010. Iexposure . Howevervia GluN2A, which might impair the high-fidelity properties of the sensory information transfer in the cerebellar GL in adult mice. Our results provide evidence for the further understanding of cellular and synaptic mechanisms of chronic ethanol exposure and their effects on motor coordination, motor learning, and cognitive functions from adolescence.Collectively, we can conclude that chronic ethanol exposure in adolescence enhances facial stimulation-evoked MF–GC synaptic transmission The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the Animal Care and Use Committee of the Yanbian University.D-LQ, B-XL, G-HD, and S-BC conceived and designed the experiments. B-XL, G-HD, H-LL, J-SZ, and Y-HB performed the experiments. C-PC and D-LQ analyzed the data. Y-HB contributed to reagents, materials, and analysis tools. D-LQ, C-PC, and S-BC wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To study the characteristics and relationship of the gut microbiota in patients with polycystic ovary syndrome (PCOS).We recruited 45 patients with PCOS and 37 healthy women from the Reproductive Department of Shengjing Hospital. We recorded their clinical indexes, and sequenced their fecal samples by 16S rDNA full-length assembly sequencing technology (16S-FAST).Prevotella enterotype when compared to control women with Prevotella enterotype, while glucose and lipid metabolism level remained no significant difference, and situations were opposite in PCOS and control women with Bacteroides enterotype. Ruminococcus gnavus, Prevotella stercorea, Dialister succinatiphilus and Bacteroides fragilis were more abundant while Christensenellaceae spp. were less abundant in the PCOS group. P. stercorea was significantly more prevalent in PCOS-not insulin resistance (NIR) compared to control-NIR and PCOS-not overweight (NOW) patient groups compared to control-NOW groups. Kyoto Encyclopedia Genes and Genomes reflecting pathways related to lipopolysaccharide biosynthesis were more abundant in the PCOS group.We found decreased α diversity and different abundances of a series of microbial species in patients with PCOS compared to healthy controls. We found LH and AMH were significantly increased in PCOS with via drugs and diet.Our study found gut microbiota that had different abundance in patients with PCOS compared to healthy controls. An intimate relationship was shown between the gut microbiota and pathological changes in PCOS. We suggest the gut microbiota should be taken into consideration in the treatment of symptoms of PCOS  Polycystic ovary syndrome (PCOS) is a reproductive endocrine disorder affecting 5%–10% of women of reproductive age . The patTen trillion bacteria exist within the human intestine, with the total number of bacterial genes about 150 times more than the number of human genes . The intBacteroides is a major genus in intestinal flora. Several species of Bacteroides are increased or decreased in number in patients with PCOS compared to healthy people in studies by Terros et al., Zeng et al., Qi et al., Liu et al., Zhang et al., and Chu et al RNA to compare the intestinal microbiome of patients with PCOS and healthy people , while shu et al . Co-abunhu et al . Insulinth SCFAs . What isth SCFAs .The term “16S” refers to 16S rDNA (or 16S rRNA). The 16S rDNA is a component of the small prokaryotic ribosomal subunit. The gene that encodes 16S rDNA has a length of about 1542 bp, including nine variable regions and 10 conserved regions. The sequence of conserved regions reflects an inter-species genetic relationship, while the variable region sequence can reflect differences between species. Traditional 16S rDNA segment sequencing technology only detects the several 16S hypervariable regions of bacteria, with the detection level only reaching the genus level. 16S rDNA full-length assembly sequencing technology (16S-FAST) can sequence full-length 16S, which provides more information than some segments, and can perform further classifications to reach a species level . Therefo2), and 12 were overweight or obese (OW) (BMI ≥24 kg/m2). Women with PCOS consisted of 14 NOW and 31 OW. Seventy-one women had their venous blood sample tested in routine clinical testing, of which 29 were controls and 42 were patients with PCOS. Twenty-two women did not have, and seven had, insulin resistance (IR) in the control group. Fifteen had PCOS without IR and 27 had PCOS with IR. This study protocol was approved by the Ethics Committee of Shengjing Hospital affiliated to China Medical University.Study participants were made up of a female population aged 18–40  who visited the outpatient clinic of the Reproductive Department of Shengjing Hospital, China Medical University. Patients were diagnosed with PCOS according to 2003 Rotterdam criteria . The incFecal samples were collected in the morning during each non-menstrual period. Venous blood samples were collected in the morning after overnight fasting (≥8 h) on the 2nd to 4th day of a spontaneous menstrual cycle, or after progestin-withdrawal bleeding. Height, weight, waist and hip circumferences were recorded on the day of fecal sampling.2), total testosterone (TT), prolactin (PRL), progestin (Prog), free triiodothyronine 3 (FT3), free triiodothyronine 4 (FT4), thyroid stimulating hormone (TSH), and fasting plasma insulin (FINS) were measured by chemiluminescence immunoassay. Anti-Müllerian hormone (AMH) was measured by enzyme immunoassay. The waist-to-hip ratio (WHR) was calculated as a waist circumference divided by a hip circumference. BMI was calculated as weight (kg) divided by height (m) squared. Free Androgen Index (FAI) was calculated as Total protein (TP), albumin (ALB), albumin/globulin (A/G), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), prealbumin (PALB), cholinesterase (CHE), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IDBIL), total bile acid (TBA), monoamine oxidase (MAO), total cholesterol (CHOL), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein A1 (apoA1), apolipoprotein B (apoB), small dense low density lipoprotein cholesterol (sd-LDL), fasting plasma glucose (FPG), sex hormone-binding globulin (SHBG) and 25-hydroxy vitamin D determination (25-(OH)VitD) were measured by enzyme-linked immunosorbent assay. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol  and then the samples were stored and sent to laboratory under room temperature. Bacterial DNA was extracted using an intestinal DNA extraction kit . Quantitative and qualitative analysis, and quality control of the extracted DNA was subsequently performed. For samples that passed quality control, 10 ng DNA was used to construct the following full-length 16S library. Full-length PCR amplification system 1 was configured, and 10 ng of DNA template was added to perform the first round of three cycles of full-length amplification. The amplified product was purified by magnetic beads.Full-length PCR amplification system 2 was nest configured, and the second round of a 32-cycle full-length amplification was performed on the product of the first round of magnetic bead purification. The full-length amplified product was then purified by magnetic beads, and the purified product was analyzed quantitatively and qualitatively.We then constructed a splicing library. Ten nanograms of full-length amplification was taken for transposase digestion. A spliced library PCR amplification system 1 was configured. Ten microlitres of the digested product was used for PCR amplification. A DNA fragment was selected from PCR-amplified products. A spliced library PCR amplification system 2 was then configured, and 2 μL of the product, after magnetic bead screening and purification, was used for the second round of a spliced library PCR amplification. The amplified product was also purified by magnetic beads. The product of this round was the spliced library.We also constructed a link library. A ligation library PCR amplification system 1 was constructed. Three nanograms was used for the first round of eight cycles of PCR amplification. The amplified product was purified by magnetic beads. Ligation library PCR amplification system 2 was configured, and 2 μL of the product of the first round of library construction and purification was used to perform the second round of eight cycles of PCR amplification. The PCR amplification product was purified by magnetic beads. The product of this round was known as the ligation library. Electrophoresis and the mesurements of Qubit concentrations were performed on spliced and ligated libraries. A library that passed quality control was sent for sequencing.U test using a scipy 1.3.1 package in python3.6. The β diversity was analyzed by an R3.6.1 package vegan2.5-3 analysis. The P value from partial least squares-discriminate analysis (PLS-DA) was calculated by matching the Adonis method. The Linear discriminant analysis Effect Size (LEfSe) was conducted by LEfSe version 1.0, parameter setting at 2, to discover gene or functional characteristics that could best explain differences between the groups. Cytoscape_v3.8.0 was used to construct a network diagram, and Spearman in SparCC was used to calculate correlation and P values. For the analysis of genus level and above, results with a correlation greater than 0.5 and P value ≤0.05 were retained; for species level, results with a correlation greater than 0.6 and P value ≤0.05 were retained. Mann–Whitney U and Kruskal–Wallis sum-rank tests of non-parametric coefficients were used to detect significant differences in clinical indexes between groups. The correlation between bacterial species and metabolic indicators was conducted by R3.6.1 package psych v1.8.4, using Spearman’s method. Heatmaps were drawn by an R package heatmap 1.0.12. PICRUSt2 was used to infer the composition of functional genes in the sample, and analyze the functional differences between different samples and groups. Python3.6 package sklearn0.23.2 was used to obtain the top 10 contribution species through recursive feature elimination, and a random forest was constructed to classify and predict samples based on flora or clinical indicators. Enterotypes were determined as follows: clustering was performed using the Center Point Partition Algorithm according to the Jensen–Shannon distance between samples and the optimal number of classifications was determined by the Calinski–Harabasz (CH) index. The cluster group with the highest CH index was considered the optimal number of groups.Unique Molecular Identifier (UMI) pairing relationships were extracted through the ligation library. All sequences corresponding to each paired UMI from the spliced library were extracted. For each paired UMI sequence, Cutadapt V1.2.1 was used to excise primers and UMI tags, and every UMI was assembled to a full-length 16S sequence through default parameters using software SPAdes V3.13.1. Mothur V1.42.0 and SILVA_132_SSURef_Nr99 databases were used for all the above sequences to perform species annotations with default parameters. The α bacterial diversity of the gut microbiota community was estimated by qiime1 V1.8.0. The difference between groups was calculated by a Mann–Whitney P < 0.05). As for biochemical indices, TP, ALB, AST, ALT, GGT, ALP, PALB, CHR, MAO, CHOL, TG, apoB, and sd-LDL were higher, while HDL-C and DBIL were lower in the PCOS group (P < 0.05). PRL, SHBG, and FSH were lower, while FAI, TT, LH, FT3, FINS, AMH, and IR were higher in the PCOS group  in the control group, which demonstrated a decrease in α diversity in PCOS women when compared to the control group though no statistical significance was reached. Beta diversity based on ASV was assessed by PLS-DA. The outcome could not separate PCOS and control groups  curve was drawn to find a model that could distinguish women with PCOS from healthy control women. The area under the curve was 0.63 when a random forest model was constructed with all species; it increased to 0.87 when the top 10 contributing species were used Figure 82 since we noticed that E2 levels of patients in PCOS-NIR and PCOS-NOW groups were lower than those of patients in control-NIR and control-NOW groups, and they showed higher level of LH than PCOS-IR and PCOS-OW, in which we hypothesized that their pituitary had a higher sensitivity to GNRH, or got less negative feedback on their pituitary due to low level of E2.We first studied the gut microbial community of PCOS patients by sequencing full-length 16S rDNA of the microbiota. In our study, participants were recruited and divided into PCOS and control groups; they were further divided into subgroups according to IR index, BMI and enterotype respectively. We analyzed clinical indexes. We noticed a trend that patients in PCOS-IR and PCOS-OW groups showed more disturbances in biochemical indexes, FINS, FPG, HOMA-IR, and TT when compared to PCOS-NIR and PCOS-NOW groups Figure 6R. gnavus, Roseburia sp. 11SE38, D. succinatiphilus, and L. bacterium 2_1_58FAA, all from the Firmicutes phylum, were increased in patients with PCOS. The increased abundance of Firmicutes is associated with a high BMI  when compared to a control group, and found to grow when IBD activity increased, which was explained as it had gene families involved in protection against the oxidative stress response in IBD gut . CD40 is positively associated with a mucosal HIV-1 viral load, and mucosal inflammatory cytokines, such as IL-23, IL-1β, IL-6, and tumor necrosis factor-α as well as with IL-10 levels. This indicates that Prevotella spp. may contribute to mucosal inflammation and the activation of immune disease  mice; the osteomyelitis-related inflammatory factor, IL-1β, was increased in LDF mice. In the former study, the Prevotellaceae family was found to be decreased in patients with PCOS, especially in the PCOS-IR group, in a study by Prevotellaceae than Bacteroidaceae, in contrast to the PCOS group. However, our data showed that the Bacteroides genus from the Bacteroidaceae family dominated in both control and PCOS groups. Twenty-six women in the control group and 31 women in the PCOS group had a Bacteroides-dominated enterotype, while only 11 women in the control group and 14 women in the PCOS group had a Prevotella-dominated enterotype. A reason for this difference may be differences in diets from northeast China (our research) and southwest China (Zeng’s research). Enterotypes are influenced by long-term dietary patterns. Eating protein-rich food, the bacteria in gut would tend to be dominated by Gram-positive bacteria while carbohydrate-rich food might lead to a Gram-negative-dominated gut environment , and super-pathway of GDP-mannose derived O antigen building block biosynthesis, which participate in the biosynthesis of a component of bacterial, lipopolysaccharide (LPS), were enriched in PCOS women  can be found below: NCBI SRA, PRJNA694729.This study protocol was approved by the Ethics Committee of Shengjing Hospital affiliated to China Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JJ and XW contributed to the conception of the study. KY performed the sequencing. ZW contributed significantly to data analyses. SD performed the data analyses and wrote the manuscript. SJ, GL, WZ, CL, and DL helped perform the analysis with constructive discussions. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No.81671423 and No.81402130).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oblique submicron-scale structures are used in various aspects of research, such as the directional characteristics of dry adhesives and wettability. Although deposition, etching, and lithography techniques are applied to fabricate oblique submicron-scale structures, these approaches have the problem of the controllability or throughput of the structures. Here, we propose a simple X-ray-lithography method, which can control the oblique angle of submicron-scale structures with areas on the centimeter scale. An X-ray mask was fabricated by gold film deposition on slanted structures. Using this mask, oblique ZEP520A photoresist structures with slopes of 20° and 10° and widths of 510 nm and 345 nm were fabricated by oblique X-ray exposure, and the possibility of polydimethylsiloxane (PDMS) molding was also confirmed. In addition, through double exposure with submicron- and micron-scale X-ray masks, dotted-line patterns were produced as an example of multiscale patterning. Nanostructured surfaces of various shapes have been studied for use in various types of research, such as for cell-behavior platforms ,2, anti-2 structures [Several of the deposition and etching techniques of lithography have been employed to fabricate oblique submicron patterns. Glancing angle deposition (GLAD) is a representative method that is used to fabricate oblique structures with various materials. After initial nucleation on the substrate, deposition proceeds, allowing nanocolumns to grow from the nuclei; the angle of the nanocolumns is determined by the deposition angle. Electron beam evaporation, which is widely used for deposition, is useful for producing SiOructures . In addiructures . Howeverructures . Plasma ructures . In contructures . Electroructures . This meructures ,21.Here, we propose a simple and angle-controllable method based on X-ray lithography to fabricate oblique submicron-scale structures with areas at the centimeter scale. Because the wavelength of X-rays is very short, diffraction is sufficiently small to form submicron-scale areal images in the photoresist; however, an oblique submicron-scale X-ray mask is not easy to fabricate. Therefore, an oblique submicron X-ray mask, which was fabricated by thermal deposition of gold on the slanted wall, was designed by improving the vertical submicron scale X-ray mask . With thTo form images in a resist, a mask is used in X-ray lithography, as in photolithography. The X-ray mask is composed of a region that is transparent to X-rays and an absorber made of heavy metal. Sufficient image contrast between the two parts must be guaranteed because this affects the patterning quality. In our system, polyimide (PI) was chosen for the transparent part due to its low absorption of X-rays and chemical stability. The absorber layer was typically several-micrometer-thick gold, as a high-Z (atomic number) material. In addition, in oblique X-ray lithography, tilted gold absorber structures are desirable for precise alignment that minimizes the transient dose region ,24. In t2. The post-bake-out process was performed for 1 min sequentially at 65 °C and 95 °C, and the development process proceeded for 1 min in a dedicated SU-8 developer . Finally, inclined micron-scale line patterns with a parallelogram shape in cross-section were formed.The negative-type epoxy photoresist SU-8 that is commonly used in UV lithography was selected as the backbone material because it shows high transmittance and durability in response to X-rays. The backbone was designed with 15-mm-long, 5-μm and 10-μm periodic 1:1 line patterns with a width of 15 mm. SU-8 3005  was spin-coated at 5000 rpm onto the PI film, which was ~4 µm thick. The soft bake-out process was performed on a hotplate at 95 °C over a period of 2 min. The mask and substrate were brought together using a vacuum and tilted 10° or 20° according to the target angle; the SU-8 photoresist was exposed to UV with a wavelength of ~365 nm at a dose of 105 mJ/cmThe absorber was deposited on the slanted backbone by thermal deposition. The geometry of the thermal deposition components determines the deposited thickness. Gold pellet  6 g was heated in a tungsten boat . The set sample loading angle, θ, of the wafer was as shown below:In X-ray lithography, higher contrast intensity is associated with more precise patterns. The contrast is calculated using the equation below:3. The double-coated PR was inclined at 20° and exposed to X-rays at a dose of 1.5 kJ/cm3 with helium. Following exposure, the development process was performed in ZED N50 developer for 30 s, followed by rinsing in the mixture of methyl isobutyl ketone and isopropyl alcohol (89:11 v/v) for 10 s.Positive ZEP 520A was selected as an X-ray resist due to its high sensitivity. ZEP 520A was spin-coated on the silicon wafer at 500 rpm and baked for 3 min at 180 °C to achieve a thickness of ~0.7 µm. In addition, one further coat of ZEP 520A was applied at a thickness of 1.5 µm for high-aspect-ratio patterns. The sample was loaded onto an aluminum jig, and the fabricated X-ray submicron mask was attached to the sample. The jig was fixed on the stage under vacuum and aligned with the X-ray angle of incidence. The single-coated PR was inclined at 10° and exposed to X-rays at a dose of 1.3 kJ/cmThe angle of the ZEP 520A photoresist was measured on cross-sectional images obtained by scanning electron microscopy . The angles of the pattern were the same as the objective angles of the mask. The line width of the 20° sample was ~510 nm, and it had an aspect ratio of ~2.94. The 80° sample had a line width of 345 nm and an aspect ratio of 2.03, as shown in w/w) and cured overnight in an oven at 60 °C . It was confirmed that the slanted PDMS groove had the same depth and period as the mold based on SEM, as shown in In addition, to fabricate groove structures, polydimethylsiloxane (PDMS) soft lithography was conducted instead of direct patterning on a negative resist. The PDMS base and the curing agent  were mixed at 10:1 (Using a combination of a nano X-ray mask and a general micron-scale X-ray mask, unique structures, including oblique dotted-dashed line structures, were manufactured. To realize such fusion structures, a typical micron-scale X-ray mask for the manufacture of micrometer-scale devices with high aspect ratios was produced using UV lithography and gold electroplating. A 100-nm-thick gold layer was deposited on a 200-μm-thick PI film as the substrate. The X-ray-transparent part was formed using negative photoresist SU-8, and the other absorber part was plated with 4-μm-thick gold . The desTo implement the fusion patterns, dotted lines were fabricated through double exposure. After the first oblique exposure using the abovementioned nano-scale X-ray mask, the second exposure using the micron-scale X-ray mask proceeded in the normal direction using the same exposure dose. Using this process, a 20° fusion pattern was fabricated, as shown in The dotted-dashed line length and spacing can be controlled by changing the fill factor of the micron-scale mask. In addition, oblique fusion structures of various shapes with different lengths or periods can be realized using this technique. As examples, short periodic line patterns were fabricated using a micron-scale mask for a 1:1 line and spacing pattern . The oblWe developed simple and angle controllable X-ray lithography method and produced oblique submicron-scale structures of ZEP 520A photoresist with areas at the centimeter scale. The engraved PDMS pattern was also produced using soft lithography. A submicron-scale X-ray mask was designed to maximize the contrast considering the beamline characteristics, and a robust thin absorber was produced by the oblique deposition method. Therefore, high-aspect-ratio 20° and 10° slanted patterns were produced that were 1.5 µm and 0.7 µm thick, respectively. Thus, the angle and thickness can be adjusted. In addition, using the double-exposure method with a micro-scale X-ray mask, the possibility of fusion patterning was confirmed for patterns including dotted lines and spacing.The developed novel method has the advantage of achieving a high aspect ratio, precise oblique angles, and a relatively large patterning area without requiring a complex system. Due to the flexibility of the X-ray mask design, pattern characteristics such as shape, length, and period can be easily controlled. This technology can be combined with other manufacturing techniques to produce hierarchical structures. Therefore, it is expected to contribute to the development of devices with asymmetric nano-scale phenomena, such as dry adhesives and hydrophobic surfaces.


In the past decade, mesenchymal stem cells (MSCs) have been widely used for the treatment of osteoarthritis (OA), and extracellular vesicles (EVs) may play a major role in the efficacy of this treatment. Hypoxia can change the cargo and biological functions of MSC-derived EVs (MSC-EVs). The aim of the present study was to determine whether the effects of hypoxia-preconditioned MSC-EVs on OA cartilage repair are superior to normoxia-preconditioned MSC-EVs. By using in vitro and in vivo OA models, we verified that hypoxia-preconditioned MSC-EVs improved chondrocyte proliferation and migration and suppressed chondrocyte apoptosis to a greater extent than normoxia-preconditioned MSC-EVs. Furthermore, we found that hypoxia altered the microRNA expression in MSC-EVs and identified four differentially expressed microRNAs: hsa-miR-181c-5p, hsa-miR-18a-3p, hsa-miR-376a-5p, and hsa-miR-337-5p. Bioinformatics analysis revealed that hypoxic pretreatment may promote cartilage repair by stimulating chondrocyte proliferation and migration and suppressing chondrocyte apoptosis through the miRNA-18-3P/JAK/STAT or miRNA-181c-5p/MAPK signaling pathway. Therefore, hypoxia-preconditioned EVs may be a novel treatment for OA. Osteoarthritis (OA) is the most common joint disease and approximately 250 million people worldwide suffer from OA; large sums of money have been invested in OA research and treatment . As a toEVs are small nanoparticles with a bimolecular membrane structure that are secreted by cells that can be found in most body fluids, such as blood, urine, and milk. Generally, according to their biological origins, EVs are mainly divided into two types: exosomes and microvesicles . EVs carIn clinical applications, many MSCs need to be expanded. However, during the expansion process, MSCs are subject to technical limitations of cell death and senescence , but it Sprague-Dawley rats that were four-weeks-old were used for this study. All the animal experimental protocols were approved by the Animal Investigation Committee of Dalian Medical University.g for 5 min. The cell pellet was resuspended after centrifugation, and the cells were plated in culture medium  and 1% penicillin-streptomycin ) at a density of 2 × 104/cm2 and incubated in 5% CO2 at 37 °C. The medium was replaced every 3 days and passage 1 (P1) cells were used for subsequent experiments.To obtain chondrocytes, the knee, hip, and shoulder joints of rats were isolated. The soft tissue around the joint was removed with ophthalmic scissors and the cartilage tissue was cut into small pieces (approximately 1 mm in diameter) and digested with 0.2% type II collagenase  for 8 h at 37 °C. The medium was filtered and collected into a centrifuge tube and then centrifuged at 1500× 2 cell culture incubator, and the adherent cells were passaged after reaching 70–80% confluence. P4 cells were used for subsequent experiments.Adult BMSCs were purchased from Cyagen Biosciences  and cultured in complete growth medium (DMEM/F-12 medium that was supplemented with 10% FBS and 1% P/S) at 37 °C in a 5% COTM V10 software.For phenotypic characterization, BMSCs were stained with mouse anti-human polyclonal antibodies against CD14, CD19, CD45, CD90, CD105, and CD73  and analyzed by flow cytometry . The data were analyzed using FlowJoFor multidirectional differentiation potential analysis, osteogenic induction medium, adipogenic induction medium, and chondrogenic induction medium  were used to replace the complete medium and induce differentiation. The process of inducing differentiation was performed as described in the manufacturer’s instructions and then assessed by Alizarin red staining, Oil Red O staining, and Alimin blue staining.2 and 21% O2 or in hypoxic cell incubators with 5% O2 in DMEM/F12 containing 10% EV-depleted FBS  for 48 h. The medium was collected and centrifuged at 3000× g for 10 min to remove the dead cells and then centrifuged at 10,000× g for 30 min to remove the cell debris. The supernatant was ultracentrifuged at 120,000× g at 4 °C for 1 h twice. The pellets were resuspended in phosphate-buffered saline (PBS) and then the protein concentration was determined [BMSCs were cultured in normal oxygen cell incubators at 37 °C with 5% COtermined . EVs werNanoSight tracking analysis  was used to measure the concentration and size distribution of EVs. Transmission electron microscopy  was used to identify EV morphology. Surface marker expression of the signature EV proteins TSG101 , CD9 , and HSP70  was determined by Western blotting.EVs were fluorescently labeled with PKH67  according to the manufacturer’s instructions. The labeled EVs were incubated with chondrocytes for 12 h. The chondrocytes were fixed with 4% paraformaldehyde, mounted using mounting medium containing DAPI , and observed under a laser confocal microscope .The Cell Counting Kit 8 (CCK-8) assay  was used to measure cell proliferation. Briefly, the chondrocytes were seeded in a 96-well plate at a density of 2000 cells/well. Next, 10 ng/mL IL-1β  was added to induce inflammation, while the control cells were not stimulated. After 24 h of modeling, the medium was replaced with fresh medium containing different concentrations of EVs . Next, 10 μL of CCK-8 solution was added and the samples were incubated for 2 h in the incubator. A microplate reader  was used to measure the optical density (OD) at a wavelength of 450 nm.4 chondrocytes were seeded into the upper chambers of a 24-well Transwell plate with 8-μm pores . Next, 600 μL of complete chondrocyte culture medium containing various concentrations of EVs was added to the lower chambers of the Transwell plate and incubated for 12 h at 37 °C. The upper chamber was then fixed with 4% paraformaldehyde for 15 min, stained with 0.5% crystal violet for 10 min and washed with PBS three times. The upper surface of the upper chamber was carefully wiped using a cotton swab to remove the cells that had not migrated to the lower surface. A total of five randomly selected fields per well were photographed using a microscope  and assessed by two pathologists in a blinded manner.A Transwell assay was used to analyze the effect of EVs that were secreted by MSCs on the migration of chondrocytes. In brief, after being digested and counted, approximately 5 × 10We used an Annexin V apoptosis detection kit  to measure apoptosis in inflammatory chondrocytes. After treatment with different concentrations of EVs for 48 h, the chondrocytes were digested, washed, and resuspended in binding buffer. Annexin V and propidium iodide (PI) were added and the samples were incubated in the dark for 15 min at room temperature. The cells that were labeled with fluorescent dyes were analyzed by flow cytometry within 30 min.p-value < 0.05 were considered to indicate a significant difference. Among miRNAs with significant differences, a fold change (FC) greater than 2 or less than 0.5 was defined as differentially expressed miRNAs. We used the GSE156919 dataset in the GEO database to verify the differentially expressed miRNAs.The extraction of tRNA from EVs, construction of an miRNA library, and miRNA sequencing were performed by LC-BIO Technologies . Standard procedures that were provided by Illumina were followed, including those for library preparation and sequencing. Small RNA sequencing libraries were prepared using TruSeq Small RNA Sample Prep Kits . After library preparation, the constructed library was sequenced using an Illumina HiSeq 2000/2500. We obtained valid data and compared precursors and genomes to identify miRNAs and performed differential analysis of miRNAs. The expression levels of miRNAs with a http://kobas.cbi.pku.edu.cn/, accessed on 15 July 2021) and DAVID 6.8  databases. KEGG pathway analysis was performed to identify the pathways that related to miRNAs and target mRNAs. GO analysis assessed the molecular functions (MFs), biological processes (BPs), and cellular components (CCs). The target genes were introduced into the STRING 11.0  database to evaluate the functional correlations between them, and, to identify hub genes, the connectivity extent in the protein-protein interaction (PPI) network was investigated through Cytoscape 3.7.1.There were two miRNA target-predicting algorithms (mirdb and TargetScan) that were used to identify the potential downstream targets of the differentially expressed miRNAs. To examine the underlying functions of the selected miRNAs and target mRNAs, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) term analysis were performed using the KOBAS 3.0 (n = 10/group): (1) Control group; (2) OA group; (3) OA + Normoxia-EV group; and (4) OA + Hypoxia-EV group. During the 4th week after surgery, the rats were injected with EVs  or an equivalent volume of PBS (100 μL). A total of eight weeks after surgery, the rats were sacrificed by anesthetic overdose and knee samples were harvested to evaluate disease progression.Female Sprague-Dawley rats (4-weeks-old) were housed in a specific pathogen-free (SPF) animal laboratory with a 12:12-h light/dark cycle, a controlled temperature environment (23–25 °C), and a steady humidity were used in these studies. The modified Hulth method was used to induce the OA model . The ratAfter being fixed with paraformaldehyde for 24 h and decalcified for 1 month in EDTA decalcifying solution , the samples were embedded in paraffin and sectioned into 5-μm-thick sections. Serial sections were obtained from the medial and lateral compartments at 200-μm intervals. The sections were dewaxed and rehydrated before the experiments. Saffron O-fast green  was used for histological staining, and the Osteoarthritis Research Society International (OARSI) scores were calculated to evaluate the degeneration of the articular cartilage. The stained sections were randomly assigned to two different pathologists for evaluation in a blinded manner.t-test was used to identify differences between groups as appropriate. A one-way analysis of variance (ANOVA) was carried out for multiple group comparisons. Statistical significance was determined at the level of p < 0.05.Each experiment was performed with at least 3 biological replicates. All data are presented as the mean ± standard deviation (SD) and were analyzed and displayed with SPSS 25.0 software . The independent P4 BMSCs were observed under an optical microscope, and the image in EVs were isolated from the BMSC supernatant by ultracentrifugation. The purified BMSC-EVs were identified by TEM, NTA, and Western blotting. TEM showed that the EVs exhibited an oval shape D. The NTTo investigate the feasibility of using BMSC-EVs to treat OA, we examined the cellular uptake of PKH67-labeled EVs. After the chondrocytes were incubated with PKH67-labeled EVs for 12 h, EVs were observed in the cytoplasm by fluorescence microscopy to confirm uptake G.Next, the effect of EVs on the chondrocytes was evaluated. IL-1β is an important factor in the development of cartilage degeneration in OA, and IL-1β was added to simulate the OA inflammatory environment. We treated inflammatory chondrocytes with different concentrations of BMSC-EVs . The CCK-8 assay results indicated that IL-1β treatment significantly reduced the proliferation of chondrocytes and EVs, especially at 20 μg/mL, and significantly attenuated the IL-1β-induced inhibition of chondrocyte proliferation A. The efTo explore the effect of hypoxia on the efficacy of BMSC-EVs in the treatment of inflammatory chondrocytes, we assessed the effect of hypoxia-EVs via CCK-8 assays, Transwell assays, and apoptosis assays. The CCK-8 assay results revealed that, compared to that of the IL-1β group, chondrocyte proliferation increased in the normoxia-EV and hypoxia-EV groups. However, hypoxia-EV administration caused a further increase in proliferation A. Next, Next, to verify the chondroprotective effect of hypoxia-EVs in vivo, a rat OA model was used. At 4 weeks after surgery, 100 μL of PBS, normoxia-EV (50 μg/100 μL), and hypoxia-EVs (50 μg/100 μL) were injected into the joint cavity. After four weeks following joint injection, we collected joint tissues for pathological evaluation. In sections that were stained with safranin O-fast green D, a redup < 0.05, a total of 29 differentially expressed miRNAs were identified in the exposure group, of which 12 miRNAs were upregulated and 17 miRNAs were downregulated , hsa-miR-18a-3p , hsa-miR-376a-5p , and hsa-miR-337-5p  were downregulated in hypoxia-EVs; a total of 913 possible target genes of these 4 miRNAs  were predicted by using mirdb and TargetScan (Based on the criteria of database C. ConsisrgetScan D.Three types of annotations for GO functions were conducted for possible target genes. The various GO functions are shown in The PPI networks of the target genes were determined using the STRING database. The data indicated that a number of target genes were able to interact with each other. The top 30 hub genes were selected based on the node degree D. ALB, SOA is a total joint disease in which the main pathological change is damage to articular cartilage. OA is characterized by anabolic and catabolic disorders of the ECM of articular cartilage. Furthermore, studies have shown that promoting chondrocyte proliferation and migration and inhibiting cell apoptosis are key points in OA treatment. Our results indicated that BMSC-EVs can promote cartilage repair through different mechanisms and that hypoxic pretreatment can enhance the therapeutic effects. The sequencing results showed that hypoxia can change the expression of miRNAs, and subsequent bioanalysis results showed that these differentially expressed miRNAs play important roles in OA.At present, many clinical studies using MSCs to treat OA have achieved certain therapeutic effects ,20. HoweThe TEM and NTA results did not show any morphological differences between the normoxic and hypoxic MSC-EV groups in terms of size, shape, or electron density. Previous studies have shown that hypoxic pretreatment increases the ability of MSCs to secrete EVs. In the present study, in vitro experiments showed that EVs promoted chondrocyte proliferation and migration and suppressed chondrocyte apoptosis and that these therapeutic effects were enhanced by hypoxic pretreatment. In the animal model of OA, the hypoxia group also showed a better therapeutic effect than the other groups.As we observed the beneficial effects of normoxia-EVs compared to hypoxia-EVs, we focused on identifying the underlying mechanisms that caused this difference between normoxia- and hypoxia-EVs. miRNAs are short RNA molecules that regulate multiple cellular processes through posttranscriptional gene silencing. Similarly, miRNAs in EVs play an important role in different BPs, and external stimuli can change the expression of miRNAs. Many studies have confirmed that hypoxia changes the expression of miRNAs, such as miR-210 ,27, miR-In total, 913 possible target genes were predicted for the four miRNAs by using miRDB and the TargetScan database. GO and KEGG enrichment analyses were performed to explore the interactions among the target genes, which were mainly enriched in BP terms that were associated with cell migration, the regulation of chondrocyte differentiation, the Wnt signaling pathway and apoptotic processes, in the MAPK signaling pathway, RAS signaling pathway, PI3K-Akt signaling pathway, and Rap1 signaling pathway. These BPs and pathways are likely to be involved in the progression of OA and cartilage repair. In the PPI network, ALB, STAT3, and MAPK1 had the highest node degrees and were selected as hub genes. Numerous studies have demonstrated that these three genes or proteins are important molecules that are associated with OA.ALB, also known as albumin, encodes the most abundant protein in human blood. ALB is the main component of synovial fluid. As OA progresses, the level of ALB also decreases. ALB has been reported to induce anti-inflammatory effects and upregulate the transcription of collagen Ⅱ during chondrogenic differentiation, thereby affecting OA treatment . CurrentOur results showed that hypoxic pretreatment could reduce the expression of miR-181c-5p, miR-18a-3p, miR-376a-5p, and miR-337-5p in EVs, and their target genes, such as ALB, STAT3, and MAPK1, are related to OA and cartilage repair. Based on these experimental results, we propose the following mechanism: hypoxia-derived EVs that are derived from BMSCs promote cartilage repair through the miRNA-18-3P/JAK-STAT or miRNA-181c-5p/MAPK signaling pathways . HoweverWe verified that BMSC-EVs promote proliferation and migration and inhibit apoptosis during cartilage repair and that hypoxic pretreatment improves these repair functions. This improvement may be due to changes in miRNA expression, and the underlying mechanism may involve the miRNA-18-3P/JAK-STAT or miRNA-181c-5p/MAPK signaling pathway. In the future, miRNAs that are combined with EVs will serve as a new strategy for the treatment of OA.